High performance liquid chromatographic assay of disaccharides and oligosaccharides produced by the digestion of glycosaminoglycans with chondroitin sulphate lyases.

Abstract

In high performance liquid chromatographic procedures hitherto described, SiO2, NH2 and RP columns have been used for the analysis of disaccharides produced by the digestion of glycosaminoglycans with the chondroitin sulphate lyases AC and ABC. The use of a potent anion exchanger offers the following advantages over these columns: superior separation characteristics for non-sulphated disaccharides, and improved column performance, coupled with more stable analytical conditions. Elution with dilute saline solutions permits separation of the two non-sulphated disaccharides from chondroitin and hyaluronate. The sequential application of chondroitinase AC and ABC permits the determination of hyaluronate, the chondroitin sulphate isomers and the dermatan sulphate isomers by high performance liquid chromatographic separation of the products of enzymatic hydrolysis. In a previously described method, hyaluronate lyase was used for the determination of hyaluronate. It has been found, however, that omission of the hyaluronate lyase step results in superior accuracy in the high performance liquid chromatographic separation of the non-sulphated disaccharides. The enzymatic analysis of human articular cartilage glycosaminoglycans has repeatedly yielded a fraction which is not digestable by chondroitinase AC, but is completely digestable by chondroitinase ABC. More extensive characterization has disclosed that this fraction differs structurally from chondroitin sulphate. Enzymatic characterization indicates that it should presumably be assigned to dermatan sulphate.